A reaction device for detecting genes, e.g. a microplate, a DNA microarray, a magnetic particle and the like (hereinafter referred to as a “reaction platform”) are available at a low price and can easily control a reaction temperature uniformly, and a special machine is not necessary for using them. Therefore, many kinds of gene diagnostic kits using a luminous enzyme or a coloring enzyme are commercially available.
At present, temperature control of these reaction platforms is performed using a usual machine called a thermostatic bath. Especially, in detecting genes, a target gene or a gene amplified by a PCR method or the like is labeled with an enzyme or the like, and the detecting reaction is usually performed as a normal biochemical reaction at a uniform reaction temperature using the thermostatic bath.
On the other hand, the present inventors have proposed a novel isothermal nucleic acid amplification method using no enzyme (a method for forming a probe self-assembly substance) (see Patent Documents 1 to 4). FIGS. 1 to 3 are schematic explanatory drawings showing the method described in Patent Document 1. For example, a method described in Patent Document 1 uses a pair of oligonucleotide probes 18 (the probe is called a “Honey Comb Probe”, and hereinafter referred to as a “HCP”) as shown in FIG. 1. The probes consist of a first HCP (a X region, a Y region and a Z region) and a second HCP (a Z′ region, a Y′ region and a X′ region). The three regions of each HCP are complementary to each other in their base sequences. The base sequences of the HCPs are designed such that they are hybridized through a couple of regions corresponding to each other when reacting them to each other (FIG. 2). By this design, when reacting plural pairs of HCPs, as shown in FIG. 9, they are hybridized with each other depending on the reaction temperature to form a self-assembly substance of probes 20 (arrows of FIG. 9) (FIG. 3). In the present specification, the method for forming a self-assembly substance by the self-assembly reaction using these oligonucleotide probes (a probe alternation link self-assembly reaction) is referred to as a PALSAR method.
Also the present inventors have found that the detection sensitivity of a target gene can be improved by using the PALSAR method (Patent Document 5). In FIG. 4, there is shown an example of a signal amplifying method with a microplate using the PALSAR method. As shown in FIG. 4(a), a capture probe 14 (a probe for capturing a gene) for a target gene 12 is bonded to the reaction device such as a microplate 10. Next, as shown in FIG. 4(b), the target gene 12 is captured. After that, as shown in FIG. 4(c), there is added oligonucleotide DNA 16 having complementary regions to a HCP and the target gene (a joint probe in Patent Document 5). In the present invention, the probe having complementary sequence to the target gene to be detected and a nucleic acid (probe) forming a self-assembly substance is referred to as an assist probe. Further, as shown in FIG. 4(e), plural pairs of HCPs 18 are added to form a self-assembly substance 20 by the self-assembly reaction, thereby the signal amplification being realized.    Patent Document 1: JP 3267576    Patent Document 2: JP 3310662    Patent Document 3: WO 02/31192    Patent Document 4: JPA 2002-355081    Patent Document 5: WO 03/029441    Patent Document 6: WO 92/20702    Non-Patent Document 1: Marshall, A., Hodgson, J. DNA chips: an array of possibilities. Nat. Biotechnol. 16, 27-31, 1998.    Non-Patent Document 2: Koshkin A A et al. Tetrahedron 1998. 54, 3607-3630.    Non-Patent Document 3: Koshkin A A et al. J. Am. Chem. Soc. 1998. 120, 13252-13253.    Non-Patent Document 4: Wahlestedt C et al. PNAS. 2000. 97, 5633-5638.    Non-Patent Document 5: H. C. Birnboim. J. Doly, Nucleic Acids Res., 7, 1513 (1979)